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Formation of the first meiotic spindle (M I) in mouse oocytes
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FORMATION OF THE FIRST MEIOTIC SPINDLE (M 1) IN MOUSE OOCYTES J. Kubiak, H. de Pennart & B. Maro Institut Jacques Monod, 2 place Jussieu, Paris, France
FREE CALCIUM RELEASE UPON FERTILIZATION IN AMPHIBIAN EGGS. Hiroshi Y. Kubota (Kyoto), Yasuaki Yoshimoto ( ~ Mit~ Yoneda ~ , and Yukio H i r a m o t o ~ , Japan). Coelomic eggs of Xenopus laevis and oviducal eggs of Cynops pyrrhogaster were preloaded with aequorin and the spatial and temporal pattern of free calcium release in the egg cortex on artificial fertilization was determined by the aequorin luminescence emitted from the eggs. The aequorin luminescence was detected with a photomultiplier and a two-dimensional photon-counting image processor. A distinct wave of free calcium release which is similar to that upon artificial activation (Develop. Biol. 119, 129-) was observed upon fertilization of Xenopus eggs. Free calcium release in Cynops eggs was also detected with a time lag on insemination and lasted for about 50 rain. The period of free calcium release which is 0.15 in a relative time scale of Cynops was comparable to 0.17 of Xenopus. However, the zone of free calcium release in Cynops eggs did not propagate as a circular wave but moved away from the sperm entrance points at a slow rate even in locally fertilized eggs. Free calcium release in uredelan eggs which show neither cortical granule exocytosis nor a visible activation wave suggests that the free calcium release upon fertilization is common in amphibians.
Mouse oocytes at the germinal vesicle (GV) stage and undergoing maturation in vitro were stained with antibodies directed against tyrosylated tubulin (YL 1/2), phopsphoproteins associated with pericentriolar material (MPM 2) and calcium sequestering system protein(s) (7/13). GV breakdown (GVBD) was monitored with anti-nuclear envelope protein, and anti-lamins. GV stage oocytes have art interphase network of microtubules (MTs), while MPM 2 only stains the GV. 7/13 antibody does not stain any structure at this stage. Disappearance of the GV, as observed with light microscopy, does not correlate with the true GVBD and nuclear lamina disassembly, which take place later. By the GVBD the network of interphase Mrs disassembles and is replaced by numerous MTs around the condensing chromosomes. MPM 2, at this stage, stains numerous, small patches located around the MTs, while 7/13 stains distinct points in direct vicinity of chromosomes 0dnetochores ?). Later, oocytes have well developed M I spindle with numerous, small, MPM 2-positive patches at the poles. During the next few hrs, MPM 2-reactive material gathers into 2-3 larger foci at each spindle pole, simultaneously with distribution of few of them into oocyte cytoplasm. The 7/13-positive antigen is detected in the kinetochore areas of forming and developed M I spindle, as well as in the cytoplasmic asters accompaning asters of MTs formed at the MPM 2-positive foci.
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Histochemistry of minced muscle a~tograft on frog g a s t r o c n e m l u s R . K . M a l h @ t r a and Rain/ Maul This p a p e r i n v o l v e s a study O f the b/stochemlcal p r o f i l e of skeletal m u s c l e during its repair and regeneration, using m i n c e d m u s c l e autografts on normal and d e n e r v a t e d frog gastrocnemil. Alterations in the levels o f glycogen and llpids have been related to changes in activities of e n z y m e s phosphorylase, llpase and succlnate dehydrogenase. Role o f ascorblc acld has also been studied. D u r l n g the e a r l y p e r i o d o f fibre dedifferentiation, the tissue m a k e s an attemt to c o n s e r v e its glycogen and l i p i d reserves w h i c h accumulate in the syncltlal matrix and the c o n n e c t i v e tissue. The three enzymes r e s p o n s i b l e for their breakdown and utilization, depict very low activities. High ascorhlc acid c o n t e n t in d e g e n e r a t i n g fibres p o i n t s t o w a r d s its role in the dedl fferentlatlon process. In the i atter phase, n e w l y dl f f e r e n t / a t e d m u s c l e fibres e x h i b i t r e d i s t r i b u t i o n of e n e r g y - r l c h fuels and the enzyme activities tend to become normal. The r a t e o f r e g e n e r a t i o n appears to be faster in the d e n e r v a t e d muscle.
Correlation between the First Cleavage Plane and the Body Axis in Xenopus laevis Embryos Rie MASHO Dept. of Polymer Science, Gunma University, Kiryu, Gunma 376 Japan One of the blastomeres of two-cellstage Xenopus laevis embryos was labeled with injected fluorescein dextran amine (FDA). The first cleavage plane was directly indicated by the boundary between the labeled and non-labeled cells in the resulting embryDs. At gastrula, neuru/a, and tail-bud stages, the topographic relation of the first cleavage plane to the dorsal midline was examined. In about 90% of the gastrulae from each batch, the first cleavage plane was found close to the dorsal midline (with the ang~!lar separation smaller than 30 degrees). Some embryos (less than 1% of each batch), howaver, had their first cleavage plane demarcate the dorsal and ventral regions. The results suggest the close relation between the first cleavage plane and the dorsal midline with some deviation. A loose but significant correlation between the pigmentation pattern and the body axis was also observed. That is, the sparsely pigmented side of the animal h~nisphere of a fertilized egg corresponds to the future dorsal region, and the densely pigmented side to the ventral region.
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439
FORMATION OF THE FIRST MEIOTIC SPINDLE (M 1) IN MOUSE OOCYTES J. Kubiak, H. de Pennart & B. Maro Institut Jacques Monod, 2 place Jussieu, Paris, France
FREE CALCIUM RELEASE UPON FERTILIZATION IN AMPHIBIAN EGGS. Hiroshi Y. Kubota (Kyoto), Yasuaki Yoshimoto ( ~ Mit~ Yoneda ~ , and Yukio H i r a m o t o ~ , Japan). Coelomic eggs of Xenopus laevis and oviducal eggs of Cynops pyrrhogaster were preloaded with aequorin and the spatial and temporal pattern of free calcium release in the egg cortex on artificial fertilization was determined by the aequorin luminescence emitted from the eggs. The aequorin luminescence was detected with a photomultiplier and a two-dimensional photon-counting image processor. A distinct wave of free calcium release which is similar to that upon artificial activation (Develop. Biol. 119, 129-) was observed upon fertilization of Xenopus eggs. Free calcium release in Cynops eggs was also detected with a time lag on insemination and lasted for about 50 rain. The period of free calcium release which is 0.15 in a relative time scale of Cynops was comparable to 0.17 of Xenopus. However, the zone of free calcium release in Cynops eggs did not propagate as a circular wave but moved away from the sperm entrance points at a slow rate even in locally fertilized eggs. Free calcium release in uredelan eggs which show neither cortical granule exocytosis nor a visible activation wave suggests that the free calcium release upon fertilization is common in amphibians.
Mouse oocytes at the germinal vesicle (GV) stage and undergoing maturation in vitro were stained with antibodies directed against tyrosylated tubulin (YL 1/2), phopsphoproteins associated with pericentriolar material (MPM 2) and calcium sequestering system protein(s) (7/13). GV breakdown (GVBD) was monitored with anti-nuclear envelope protein, and anti-lamins. GV stage oocytes have art interphase network of microtubules (MTs), while MPM 2 only stains the GV. 7/13 antibody does not stain any structure at this stage. Disappearance of the GV, as observed with light microscopy, does not correlate with the true GVBD and nuclear lamina disassembly, which take place later. By the GVBD the network of interphase Mrs disassembles and is replaced by numerous MTs around the condensing chromosomes. MPM 2, at this stage, stains numerous, small patches located around the MTs, while 7/13 stains distinct points in direct vicinity of chromosomes 0dnetochores ?). Later, oocytes have well developed M I spindle with numerous, small, MPM 2-positive patches at the poles. During the next few hrs, MPM 2-reactive material gathers into 2-3 larger foci at each spindle pole, simultaneously with distribution of few of them into oocyte cytoplasm. The 7/13-positive antigen is detected in the kinetochore areas of forming and developed M I spindle, as well as in the cytoplasmic asters accompaning asters of MTs formed at the MPM 2-positive foci.
440
441
Histochemistry of minced muscle a~tograft on frog g a s t r o c n e m l u s R . K . M a l h @ t r a and Rain/ Maul This p a p e r i n v o l v e s a study O f the b/stochemlcal p r o f i l e of skeletal m u s c l e during its repair and regeneration, using m i n c e d m u s c l e autografts on normal and d e n e r v a t e d frog gastrocnemil. Alterations in the levels o f glycogen and llpids have been related to changes in activities of e n z y m e s phosphorylase, llpase and succlnate dehydrogenase. Role o f ascorblc acld has also been studied. D u r l n g the e a r l y p e r i o d o f fibre dedifferentiation, the tissue m a k e s an attemt to c o n s e r v e its glycogen and l i p i d reserves w h i c h accumulate in the syncltlal matrix and the c o n n e c t i v e tissue. The three enzymes r e s p o n s i b l e for their breakdown and utilization, depict very low activities. High ascorhlc acid c o n t e n t in d e g e n e r a t i n g fibres p o i n t s t o w a r d s its role in the dedl fferentlatlon process. In the i atter phase, n e w l y dl f f e r e n t / a t e d m u s c l e fibres e x h i b i t r e d i s t r i b u t i o n of e n e r g y - r l c h fuels and the enzyme activities tend to become normal. The r a t e o f r e g e n e r a t i o n appears to be faster in the d e n e r v a t e d muscle.
Correlation between the First Cleavage Plane and the Body Axis in Xenopus laevis Embryos Rie MASHO Dept. of Polymer Science, Gunma University, Kiryu, Gunma 376 Japan One of the blastomeres of two-cellstage Xenopus laevis embryos was labeled with injected fluorescein dextran amine (FDA). The first cleavage plane was directly indicated by the boundary between the labeled and non-labeled cells in the resulting embryDs. At gastrula, neuru/a, and tail-bud stages, the topographic relation of the first cleavage plane to the dorsal midline was examined. In about 90% of the gastrulae from each batch, the first cleavage plane was found close to the dorsal midline (with the ang~!lar separation smaller than 30 degrees). Some embryos (less than 1% of each batch), howaver, had their first cleavage plane demarcate the dorsal and ventral regions. The results suggest the close relation between the first cleavage plane and the dorsal midline with some deviation. A loose but significant correlation between the pigmentation pattern and the body axis was also observed. That is, the sparsely pigmented side of the animal h~nisphere of a fertilized egg corresponds to the future dorsal region, and the densely pigmented side to the ventral region.
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